The present invention relates to novel thrombin inhibitors and, in particular, thrombin inhibitors derived from leech tissue and leech secretions.
Thrombin catalyzes the formation of fibrin clots and inhibits, therefore, the coagulation of blood. Moreover, thrombin has several other bioregulatory roles such as the direct activation of platelet aggregation and the activation of inflammatory response by stimulating the synthesis of platelet activating factor (PAF) by endothelial cells. That means that thrombin plays a central role in thrombosis related disorders such as cardiovascular desease, for example.
Consequently, there is a great interest in a continual search for new or improved thrombin inhibotors and anticoagulants, respectively.
Examples of well known thrombin inhibitors are heparin and hirudin.
Heparin accelerates the anticoagulant activity of antithrombin III. It has been widely used to treat conditions, such as venous thromboembolism, in which thrombin activity is responsible for the development or expansion of a thrombus. It is not effective for therapy in the cases where antithrombin III is decreased, for example in the cases of thrombosis associated with nephrosis or disseminated intravascular coagulation syndrome (DIC). Moreover, heparin produces many undesirable side effects, including hemorrhaging and thrombocytopenia.
Hirudin is a well known and well characterized polypeptide, which is known to be specific for thrombin, and which may be isolated from extracts of the salivary gland and other tissues of leeches of the species Hirudo medicinalis. Hirudin and its derivatives are also obtainable by recombinant techniques. The polypeptide has a relatively low molecular weight of 7000 D and is comprised of 65 amino acids. The amino acid sequence of hirudin was first determined by Dodt et al. (FEBS Letters, 165, 180-184, 1984). Three major variants of hirudin (HV1, HV2, HV3) have been found in the medicinal leech Hirudo medicinalis and differ in only about 10% of the total amino acid positions. The most notable difference is at the first two positions of the N-terminal end of the molecule: Val-Val in hirudin variant 1 (HV1) and Ile-Thr in hirudin variant 2 (HV2). These differences are of minor nature and do not affect the function nor the specificity of hirudin-thrombin interaction. Hirudin is a potent natural inhibitor of coagulation. It has demonstrated efficacy in preventing venous thrombosis, vascular shunt occlusion and thrombin-induced disseminated intravascular coagulation, but shows, however, prolonged bleeding times.
Phylogenetically, the medicinal leech Hirudo medicinalis is a member of the sub-family Hirudininae of the leech familiy Hirudinidae (R. T. Sawyer: "Leech Biology and Behaviour", Oxford University Press, Vol. 2, p.688, 1986). An evolutionarily more advanced leech species is Hirudinaria manillensis, which belongs to the sub-family Hirudinariinae of the same family Hirudinidae. Unexpectedly, a related but distinctly quite different isoform of hirudin was discovered recently in Hirudinaria manillensis as described in PCT patent application WO 90/05143. This isoform differs in nearly 40% of the amino acid positions when compared with hirudin from Hirudo medicinalis. The two species mentioned above, namely Hirudo medicinalis and Hirudinaria manillensis, belong to the leech order Arhynchobdellida ("jawed leeches"). In addition to the order Arhynchobdellida, there is one other major order of leeches, i.e. Rhynchobdellida ("proboscis leeches") (R. T. Sawyer: "Leech Biology and Behaviour", Oxford University Press, Vol. 2, p.651, 1986). The best studied member of the order Rhynchobdellida, with respect to salivary proteins, is the "Amazon leech", Haementeria ghilianii.
It has been demonstrated that this species, unexpectedly, does not contain an antithrombin (Budzynski et al.: Proc. Soc. Exp. Biol. Med, 168, 259-265, 1981 ). Instead, Haementeria ghilianii contains a fibrinogenolytic enzyme called Hementin (US Patent 4390630), as well as an inhibitor of the blood coagulation factor Xa (C. Condra et al.: Thromb. Haemost., 61,437-441, 1986).
Based on this discovery and subsequent work, it has become generally accepted that antithrombin-like activity is restricted to leeches of the order Arhynchobdellida, whereas antithrombin-like activity is currently thought to be lacking in the order Rhynchobdellida.
Despite the developments discussed above, the need continues to exist for supplying besides heparin and hirudin further anticoagulants and antithrombins, respectively, having increased efficacy in the inhibition of clot formation, thrombin-induced platelet activation or endothelial cell activation, which may be produced in commercially feasible quantities.